Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Less than 5% of patients survive more than 2 years and new therapeutic strategies are desperately needed. The Notch signaling pathway plays a crucial role in regulation of multi-potent stem cells in the development of the human central nervous system. In addition, it has been shown that GBMs contain a sub-population of cancer stem-like cells (CSLCs) that demonstrate elevated Notch activity. However, it is as yet unknown whether Notch pathway blockade could target CSLCs in GBM. Recent studies have shown that primary cultures of GBM propagating as neurospheres more accurately replicate the infiltrating growth patterns seen in the native brain tumor, and contain CSLCs with stem-like multi-potent capacity. Among solid tumors, brain CSLCs are by far the mostly well-studied. This is due, at least in part, to the well-developed neurosphere culture systems. We have obtained four GBM neurosphere lines from Dr. Angelo Vescovi. Other GBM models we study include several lines of primary patient-derived GBMs engrafted intracranially in immunocompromised mice obtained from Dr. Sean Morrison, as well as three primary low-passage GBM cultures. We are, therefore, well positioned to further address this exciting new area of cancer biology. In the current application, we propose a series of studies using these models, focusing on targeting GBM CSLCs by Notch pathway inhibition (gamma secretase inhibitor-18), both in vitro and in vivo. We will also design a realistic combination therapeutic regimen to facilitate its clinical translation. Specific Aim #1 is to determine effects of Notch pathway inhibition on CSLCs in primary GBMs in vitro and in vivo. Specific Aim #2 is to examine the effects of Notch inhibition in combination with Akt inhibition or common chemo-radiation therapies on GBM CSLC-derived mouse tumor model. Success in the current proposal will not only enhance our understanding CSLC biology in general, but also has the potential for direct translational impact on GBM treatment in our patients.